Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

Pre-existing data:
There are no pre-existing data available that would address the developmental toxicity endpoint (GLP or non-GLP). There are no historical human data that could be used in place of this study.

- (Q)SAR
There are no accepted QSAR approaches for predicting the outcome of this endpoint.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
There are no data on the registered substance itself.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint.

- Substance-tailored exposure driven testing
To date, exposure-based waiving of this endpoint has not been accepted by the Agency. It is almost impossible to formulate a sufficiently robust case for exposure based waiving due to the need to demonstrate 'No' exposure

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The arguments presented in this proposal demonstrate that there are inadequate alternative approaches and waving justifications to address the information requirement.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: dark red/brown liquid
- Analytical purity: 96.09%
- Lot/batch No.: S
- Stability under test conditions: the test material was stated to react with strong oxidizing agents. According to the study report, it was exposed to no such materials, with the exceptions of those potenially present in the testing environment.
Specific details on test material used for the study:
Identification: N,N’-Di-sec-butyl-p-phenylenediamine
Appearance: Red/Brown oily liquid
Batch: W8A18GN001
Purity/Composition: See certificate of analysis (see Appendix 4)
Test item storage: In refrigerator (2-8°C) protected from light container flushed with nitrogen
Stable under storage conditions until: 22 January 2019 (expiry date)

Test animals

Species:
rat
Strain:
other: Wister Han
Details on test animals and environmental conditions:
On 05 Jun 2018 (first 11 animals: Subgroups 1 and 2) and 22 Jun 2018 (second 11 animals: Subgroups 3 and 4), time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). Animals weighed between 184 and 256 g at the initiation of dosing. A health inspection was performed upon receipt of the animals.

Justification for Test System and Number of Animals
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified by indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
One day after receipt (Subgroups 1 and 2) or on the day of receipt (Subgroups 3 and 4), animals were assigned to groups by a computer-generated random algorithm according to body weights. Each set of females mated on the same date (i.e. 4 sets) was distributed as evenly as possible over the dose groups with body weights within ± 20% of the mean for each set of animals.

Housing
On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The daily average temperature during the study period was between 20 to 21°C with an actual daily mean relative humidity of 40 to 77% (see deviations in Appendix 7). A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 24 hours after adding the vehicle to the test item. Details of the preparation and dispensing of the test item have been retained in the Study Records. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

Administration of Test Materials
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 20 post-coitum, inclusive. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20140513 ).

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory . Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ~ 10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20140513) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20140513.
Details on mating procedure:
Time-mated females were used on the study, so this does not apply.
Duration of treatment / exposure:
Gestation day 6-20
Frequency of treatment:
Daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
0 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were treated with the test article from gestation days 6-20, at which time a caesarian section was performed. Dams were given a gross necropsy, and the fetuses were given a full gross examination, fresh visceral examination and skeletal exam for variations and malformations

Examinations

Maternal examinations:
Mortality/Moribundity Checks – F0-Generation
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations – F0-Generation
Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy. For animals from Subgroup 3, clinical observations started on Day 3 post-coitum

The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect premature birth.

Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. For animal no. 88, the first body weight value was recorded on Day 3 post-coitum. In order to monitor the health status, animal no. 88 was also weighed on Day 14 post-coitum.

Food Consumption – F0-Generation Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

Water Consumption – F0-Generation Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Ovaries and uterine content:
All animals (including females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals that delivered early).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
• The sex of each fetus based on the ano-genital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
Fetal examinations:
Fetal Examinations (scheduled) – F1-Generation
Litters of females surviving to scheduled necropsy, or that delivered on the day of scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation
Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. A visceral and skeletal examination was also performed for the nonviable fetuses (see deviations in Appendix 7).

Visceral Examinations– F1-Generation
The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe ref 1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar ref 2.
The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique ref 3. After examination, the tissues without variation or malformations were discarded .
All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations– F1-Generation
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson ref 4.
Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads ).
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or % CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

on-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test item-related clinical signs were observed in all treated groups:
Persistent green discoloration of the urine was observed among all treatment groups starting at 10 mg/kg. This clinical sign was noted in all females at 30 and 60 mg/kg, after 1-2 days of treatment (from Day 7-8 post-coitum onwards) and in 15 out of 22 animals at 10 mg/kg, on average starting during the second week of treatment (for animals nos. 23, 26, 27, 28, 29, 30, 32, 33, 40, 41, 43 and 44 starting on Days 13-19 post-coitum and for animals nos. 24, 31 and 42 starting on Days 7-11 post-coitum).

Piloerection was observed for several consecutive days in 4 out of 22 animals at 60 mg/kg (nos. 80, 81, 82 and 88), with two of them (no. 80 and 88) presenting with hunched posture for 2 and 10 consecutive days, respectively. Animal no. 88 was also noted with reflux on one single day (Day 13 post-coitum), flat gait for 8 consecutive days, slight and transient laboured respiration and rales for 1-3 days together with slight pale ears and ptosis for 1-2 days.

Salivation noted in one control, 2 animals at 10 mg/kg and among 11 and 10 animals at 30 and 60 mg/kg, respectively was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Other clinical signs observed during the treatment period included slight rales in one mid-dose animal on one single day and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and were not considered to be toxicologically relevant.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg, body weight gain was statistically significantly lower than control mean on Day 9 post-coitum. This decrease in body weight gain was mainly attributed to 6 high-dose females (nos. 68, 72, 78, 80, 83 and 88) that showed a body weight loss of 1-9% or absent body weight gain over Days 6-9 post-coitum, followed by a recovery afterwards (except for female no. 80 that was noted with low body weight gain during the entire study period as presented with only early resorptions). From Day 12 post-coitum until the end of the treatment period, mean body weight gain of animals at 60 mg/kg returned back to normal values. Mean body weights at 60 mg/kg remained in the same range as controls over the study period.
Body weight and body weight gain at 10 and 30 mg/kg was considered to be unaffected by treatment.
No toxicologically relevant changes in body weight gain corrected for gravid uterus were noted by treatment up to 60 mg/kg. The statistically significant increase in body weight corrected for gravid uterus at 30 mg/kg was considered to be unrelated to treatment since no trend was apparent regarding dose and since mean values were within the historical control range .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg, body weight gain was statistically significantly lower than control mean on Day 9 post-coitum. This decrease in body weight gain was mainly attributed to 6 high-dose females (nos. 68, 72, 78, 80, 83 and 88) that showed a body weight loss of 1-9% or absent body weight gain over Days 6-9 post-coitum, followed by a recovery afterwards (except for female no. 80 that was noted with low body weight gain during the entire study period as presented with only early resorptions). From Day 12 post-coitum until the end of the treatment period, mean body weight gain of animals at 60 mg/kg returned back to normal values. Mean body weights at 60 mg/kg remained in the same range as controls over the study period.

Body weight and body weight gain at 10 and 30 mg/kg was considered to be unaffected by treatment.

No toxicologically relevant changes in body weight gain corrected for gravid uterus were noted by treatment up to 60 mg/kg. The statistically significant increase in body weight corrected for gravid uterus at 30 mg/kg was considered to be unrelated to treatment since no trend was apparent regarding dose and since mean values were within the historical control range .
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A test item-related increase in mean liver weight and liver/body weight ratio was observed at a dose-related severity . Relative mean liver weights were around 23% and 37% higher than concurrent control mean at 30 and 60 mg/kg, respectively. Higher liver weights were also observed in a previously performed Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar Han rats with N,N’-Di-sec-butyl-p-phenylenediamine (Test Facility Study no. 20140515) in which non-adverse test item-related microscopic alterations were recorded in the liver of females treated at 60 mg/kg for 28 days, consisting of hepatocellular hypertrophy and/or increased cytoplasmic rarefaction, with correlating increased relative liver weight (27%).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment up to 60 mg/kg .
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to concurrent control mean (9.3 vs 11.4). This was considered to be related to the higher incidence of early resorptions (caused by 2 litters; each consisting of 9 and 12 early resorptions), resulting in a higher litter incidence of post-implantation loss at 60 mg/kg. The higher number of early resorptions observed in these two high-dose females could be regarded as secondary to maternal toxicity (body weight loss over Days 6-9 post-coitum) and as such not considered a direct effect of the test item on fetal development
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to concurrent control mean (9.3 vs 11.4). This was considered to be related to the higher incidence of early resorptions (caused by 2 litters; each consisting of 9 and 12 early resorptions), resulting in a higher litter incidence of post-implantation loss at 60 mg/kg. The higher number of early resorptions observed in these two high-dose females could be regarded as secondary to maternal toxicity (body weight loss over Days 6-9 post-coitum) and as such not considered a direct effect of the test item on fetal development
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean fetal body weights were higher in both sexes at 30 and 60 mg/kg when compared to concurrent controls. Mean combined weights were 8% and 6% significantly higher than control mean, respectively. Mean values were also higher than the 95th percentile of the historical control data (combined weight P95=5.4 gram).
No statistically significant changes in fetal body weight (both sexes) were noted at 10 mg/kg.
Mean combined (male and female) fetal body weights were 5.2, 5.4, 5.6 and 5.5 gram for the control, 10, 30 and 60 mg/kg groups, respectively.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to controls (9.3 vs 11.4). This was considered to be related to a higher incidence of early resorptions (caused by litter A078 and A080; each consisting of 9 and 12 early resorptions). The higher number of early resorptions observed in these two high-dose females could be related to their body weight loss over Days 6-9 post-coitum and, therefore, was regarded as secondary to maternal toxicity and as such not considered a direct effect of the test item on fetal development.
Mean litter sizes were 11.4, 10.2, 10.6 and 9.3 fetuses/litter for the control, 10, 30 and 60 mg/kg groups, respectively.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to controls (9.3 vs 11.4). This was considered to be related to a higher incidence of early resorptions (caused by litter A078 and A080; each consisting of 9 and 12 early resorptions). The higher number of early resorptions observed in these two high-dose females could be related to their body weight loss over Days 6-9 post-coitum and, therefore, was regarded as secondary to maternal toxicity and as such not considered a direct effect of the test item on fetal development.
Mean litter sizes were 11.4, 10.2, 10.6 and 9.3 fetuses/litter for the control, 10, 30 and 60 mg/kg groups, respectively.


Mean fetal body weights were higher in both sexes at 30 and 60 mg/kg when compared to concurrent controls. Mean combined weights were 8% and 6% significantly higher than control mean, respectively. Mean values were also higher than the 95th percentile of the historical control data (combined weight P95=5.4 gram).
No statistically significant changes in fetal body weight (both sexes) were noted at 10 mg/kg.
Mean combined (male and female) fetal body weights were 5.2, 5.4, 5.6 and 5.5 gram for the control, 10, 30 and 60 mg/kg groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on external morphology following treatment up to 60 mg/kg.
Two externally malformed fetuses were observed in this study: snout proboscis was noted in one control fetus (A002-03) and one fetus at 10 mg/kg (A037-03) was noted with a small lower jaw. Both findings were skeletally confirmed. Besides, during evisceration of this low-dose fetus (A037-03) prior to skeletal staining, the visceral malformation absent eye was also noted. At the incidence observed (occurrence in a single fetus), presence only in the control group and absence of a dose-response relationship, these malformations were considered to be of spontaneous origin and, therefore, unrelated to treatment with the test item.
No external variations were noted in any of the groups
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal malformations were noted by treatment up to 60 mg/kg.
With regard to the observed skeletal variations, there was a treatment-related decrease in bent ribs at 30 and 60 mg/kg. Incidences were 22.5%, 21.7%, 11.4% and 5.9% in the control, 10, 30 and 60 mg/kg groups, respectively (reaching statistical significance in the high-dose group). The decreased incidence of bent ribs could be attributed to the higher mean fetal body weight observed in the mid- and high-dose groups. This finding was not considered to be toxicologically relevant as incidences were within the historical control range (mean=14.0%, P5-P95=1.8-26.1%) and since a lower incidence for bent ribs is not considered to have any detrimental effect.
Any other skeletal variations occurred in the absence of a dose-related trend, low occurrence (i.e. in one single fetus) and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered to be treatment related.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on visceral morphology following treatment up to 60 mg.
Two viscerally malformed fetuses were observed in the current study: one control fetus was noted with a small eye (A012-08) and one fetus at 10 mg/kg (A037-03) presented with an absent eye. Both findings were skeletally confirmed and were considered to have occurred by chance, at the incidence observed and in the absence of a dose-response relationship.
Only one visceral variation (small supernumerary liver lobes) was observed in the control and all treated groups. At the low incidence observed (well within the historical control range) and in the absence of a dose-response relationship, this finding was not considered to be related to treatment. Other observed visceral variations (discoloured liver and convoluted ureter) were only present in the control group and, therefore, considered not to be test item-related.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Early resorptions

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg .
The developmental NOAEL for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg.
Executive summary:

The objectives of this study were to determine the potential of N,N’-Di-sec-butyl-p-phenylenediamine to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 10, 30, 60 mg/kg/day, based on the results of the dose range finder (Test Facility Study No. 20140512)

 

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, organ weights (liver), number of corpora lutea, (gravid) uterine weight and uterine contents.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

No mortality occurred during the study period.

Test item-related clinical signs were observed in all treated groups:

Persistent green discoloration of the urine was observed among all treatment groups starting at 10 mg/kg. This clinical sign was noted in all treated animals at 30 and 60 mg/kg, after 1-2 days of treatment (from Day 7-8 post-coitum onwards) and in 15 out of 22 animals at 10 mg/kg, on average starting during the second week of treatment. Although no clear cause could be attributed to this finding, it was considered not to be toxicologically relevant since clear signs of systemic toxicity would have been observed in case of renal toxicity among animals presenting with this abnormal colour in urine (no relevant clinical signs were observed in any of the Group 2 and 3 animals and in 18 out of 22 animals in Group 4 presenting with green urine).

At 60 mg/kg, 4 out of 22 animals were noted with hunched posture and/or piloerection for several consecutive days, with one of them additionally presenting with reflux on one single day (Day 13 post-coitum), flat gait for 8 consecutive days, slight and transient laboured respiration and rales for 1-3 days together with slight pale ears and ptosis for 1-2 days.

At 60 mg/kg, mean body weight gain and food consumption were statistically significantly lower than control mean during the first days of treatment (over Days 6-9 post-coitum), followed by a recovery afterwards. As complete recovery was noted, the transient decrease in mean body weight gain and food consumption was not considered adverse. From Day 12-15 post-coitum onwards a significant increase in food consumption was observed at 60 mg/kg (15-16%vscontrol), that might have occurred to compensate for the initial decrease in food intake observed during the first days of the treatment period.

No toxicologically relevant changes in mean body weight, body weight gain and food consumption were observed at 10 and 30 mg/kg over the entire study period. The slight increase in food consumption (<10%vscontrol) observed at 30 mg/kg over Days 12-21 post-coitum was considered non-adverse.

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment up to, and including, 60 mg/kg.

A test item-related increase inmean liver weight and liver/body weight ratiowas observed in a dose-response manner. Relative mean liver weights were 23% and 37% higher than concurrent control mean at 30 and 60 mg/kg, respectively. In a previously performed Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar Han rats with N,N’-Di-sec-butyl-p-phenylenediamine (Test Facility Study no. 20140515) a test item-related increase in mean relative liver weight was also observed in females treated at 30 and 60 mg/kg for at least 28 days (15% and 27%vscontrol, respectively), together with incidental/background hepatic microscopic findings at 30 mg/kg and non-adverse test item-related microscopic alterations at 60 mg/kg (hepatocellular hypertrophy with increased incidence and/or severity of cytoplasmic rarefaction). However, thepossible adversity of thedose-dependent moderate increase in liver weightobserved in the current study was not assessed histopathologically and was, therefore, not taken into account when determining the maternal NOAEL.

The number of pregnant females, corpora lutea, implantation sites, and pre-implantation loss in the control and treatment groups was in the range of normal biological variation.

There were 2 females, one each at 30 and 60 mg/kg that had an early delivery on the day of scheduled necropsy (1 viable pup + 11 viable fetuses and 4 viable pups + 10 dead fetuses, respectively). Although this number of fetal deaths was noticed in a female that delivered early on the day of schedule necropsy (Day 21 post-coitum), a relationship to treatment could not be completely discarded as it occurred in the high dose group.

At 60 mg/kg, mean number of viable fetuses was statistically significantly lower when compared to concurrent control mean (9.3vs11.4). The % per litter of viable fetuses in this high dose group was also lower when compared with concurrent control mean (89.1%vs97.5%) and the 5thpercentile of the historical control data (90.8%). This was considered to be related to the higher incidence of early resorptions (caused by 2 litters; each consisting of 9 and 12 early resorptions), resulting in a higher litter incidence of post-implantation loss at 60 mg/kg. While maternal toxicity may have played a role in the increase in early resorptions in the two affected dams in the 60 mg/kg group, it was not possible to assign a cause and effect relationship nor was it possible to rule out a direct effect of the test item on the survival of the embryo.

Mean combined fetal body weights were 8% and 6% highervscontrol mean at 30 and 60 mg/kg, respectively, and values were outside the historical control range. The increase in mean fetal body weights in both sexes could partly be explained by the slightly higher mean food consumption (absolute and relative) noted in females at 30 and 60 mg/kg from Day 12-15 post-coitum onwards. This observation in fetal body weight was considered not to be of toxicologically relevance due to the slight magnitude of the change (less than 10%), absence of test item-related malformations or detrimental developmental variations and as the opposite effect (i.e. a decrease) would be expected in case of intrauterine/fetal growth retardation.

No statistically significant changes in fetal body weights (both sexes) were noted at 10 mg/kg.

The treatment-related decrease in the skeletal variation bent ribs observed at 30 and 60 mg/kg could be related by the higherfetal body weightobserved in both groups. This variation was considered not to be toxicologically relevant as incidences were within the historical control range and since a lower incidence for bent ribs is not considered to have any detrimental effect.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. sex ratio, external and visceral malformations and developmental variations, and skeletal malformations).

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg (based on clinical signs at 60 mg/kg).

The developmental NOAEL for N,N’-Di-sec-butyl-p-phenylenediamine was established as being 30 mg/kg (due to the higher incidence of early resorptions in 2 females at 60 mg/kg).